Exercise 3

Gram Staining

1. Give the Gram-staining reaction and morphology (shape) of the following

bacteria.

Gram stain reaction: Gram-positive; turns blue

Morphology/Shape: Cocci (Spheres)

Gram stain reaction:

Gram-positive; turns purple
Gram-negative; turns pink
Morphology/Shape: Bacilli (Rods)

Gram stain reaction: Gram-negative; turns

pink/red

Morphology/Shape: Bacilli (Rods)

2. Indicate the function of the different reagents used in Gram-staining

Reagent Function Expected Result

Gram positive Gram negative
Crystal violet Primary stain Purple or blue Purple or blue
Gram’s iodine Mordant Purple or blue Purple or blue
95% Alcohol Decolorizer Purple or blue Colorless
Safranin Counterstain Purple or blue Red or pink
(Secondary
stain)

3. Differentiate gram-positive cell wall from gram-negative cell wall.

Features Gram-positive cell Gram-negative cell

wall wall
Peptidoglycan Thick Peptidoglycan Thinner
Multi-layered Peptidoglycan
Single layered
Complexity - Less complex - More complex
Teichoic Acid - Can elicit - None
antibody
response
- Attachment of
the organisms
to the host cell
- Provide tensile
strength
- Serve as pores
allowing the
passage of ions,
nutrients, and
 amino acids
into the cell
- Serve as
adhesins,
allowing
bacteria to bind
to one another
Lipopolysaccharide - Gram-positive - It is a highly
complexes bacteria do not acylated
contain LPS, saccharolipid
but carry located on the
surface teichoic outer leaflet of
acids, the outer
lipoteichoic membrane of
acids and Gram-negative
peptidoglycan bacteria
instead. - It is critical to
maintaining the
barrier function
preventing the
passive
diffusion of
hydrophobic
solutes such as
antibiotics and
detergents into
the cell
Endotoxin - As gram- - Endotoxins are
positive also known as
bacteria lack an lipopolysacchar
outer ides and are
membrane, they found in the
do not produce outer
endotoxins. membranes of
gram-negative
bacteria. They
elicit a strong
immune
 response in man
(e.g., fever,
septic shock),
and cannot be
removed from
materials by
normal
sterilization
processes.
- Endotoxins are
integral
components of
the outer
membrane of
gram-negative
bacteria such as
Salmonella,
Shigella and
Escherichia
coli. The
specific
component
responsible for
endotoxin
activity of this
bacteria is the
lipopolysacchar
ide (LPS),
which is further
composed of a
lipid A moiety
and a
polysaccharide
moiety